1. Field of the Invention
The present invention provides a novel methods for treating disorders and diseases which have an underlying or associated neurogenic component, e.g. inflammatory diseases such as arthritis, in order to block or suppress inflammation leading to the unpleasant side effects of neuropathic pain associated with the underlying or associated inflammatory response in these diseases/disorders. The present invention provides a method using Clostridium botulinum toxins alone or in combination with other serotypes of botulinum toxin or other related toxins, fugl proteins or neuropeptides, to antagonize the release or enzymati, cleave neuropeptides, neurotransmitters and other mediators from, in particular, sensory afferent or efferent neurons, autonomic efferent nerves or secretory cells. In addition, cells that release neuropeptides and other mediators, activators or promoters of inflammation such as sensory and autonomic neurons and other secretory cells play a role in inflammation. Botulinum toxins block the actions of these mediators by acting enzymatically via the metallopeptidase or related activity associated with the toxins, to cleave peptides critical to normal vesicular secretion and/or proteolytically cleave the peptide mediators
2. Description of Prior Art
Botulinum toxin (BoNT) is a paralytic neurotoxin that has become widely investigated for its therapeutic potential in the treatment of a variety of neuro-muscular disorders including: blepharospasm, spasmodic dysphonia, Strabismus, hemifacial spasm, and adult onset spasmodic torticollis. (Simpson, 1981; Habermann, 1989; Jankovic and Brin, 1991; Borodic et al., 1991; Hambleton, 1992; Schantz and Johnson, 1992; Valtorta and Arslan 1993). Intramuscular injection of nanogram quantities of purified botulinum toxin results in the toxin binding to presynaptic cholinergic nerve terminals and inhibits the release of acetylcholine and thus decreases muscle activity. The result is relaxation of the tonically contracted muscle and thus relaxation of the intended muscle or muscle group.
Botulinum toxin from the Clostridial species is a common term applied, to date to seven immunologically distinct serotypes referred to as A, B, C, D, E, F, and G (Jankovic and Brin, 1990). BoNTs are composed of a heavy chain (MW=100,000Da) and a light chain (MW=50,000Da) joined by disulfide bonds. The light chain is thought to possess a zinc-dependent endopeptidase activity (a metalloendopeptidase) responsible for the cleavage of neuronal proteins. BoNT types A and E cleave the protein SNAP-25, (Blasi et al., 1993a; Binz et al., 1994; Schiavo et al., 1993a,b), BoNT type G cleaves the protein VAMP/synaptobrevin (Yamasaki, 1994). Specifically the B, D, and F toxins cleave VAMP-2/synaptobrevin-2 isoform (Schiavo et al., 1992,1993a,b), and the D and F toxins cleave the VAMP-1/synaptobrevin-1 isoform. BoNT type C.sub.1 toxin selectively cleaves HPC-1/syntaxin, in particular, syntaxin 1A and 1B isoforms (Blasi et al., 1993b; Montecucco and Schiavo, 1994; Schiavo et al. 1995). Two laboratories recently reported that BoNT type C.sub.1 cleaves not only syntaxin but also has an affinity to cleave the carboxyl terminus of SNAP-25 when studied in chromaffin cells and spinal cord neurons (Foran et al, 1996, Williamson et al., 1996). Proteolytic cleavage of these specific proteins within nerve terminals can result in a profound decrease in normal neurotransmitter release and ultimately the hallmark signs of botulism; diffuse muscular paralysis, impaired vision, and paralysis of the diaphragm leading to suffocation and eventual death. (Donadio et al., 1971; Hughes et al., 1981).
Botulinum toxin is obtained commercially by establishing and growing cultures of C. botulinum in a fermenter and harvesting and purifying the fermented mixture in accordance with known techniques.
Botulinum toxin type A is commercially available for the treatment of the above disorders by Allergan, Inc., Irvine, Calif. under the trade name BOTOX.RTM. and from Ipson Boufor, France, under the trade name Dysport.RTM..
The contribution of the nervous system in inflammation has been recognized since Lewis (1932, 1936) proposed that the characteristic wheal and flare responses are mediated by the release of pro-inflammatory substances (described in detail below) from peripheral nerve endings of nociceptive afferent pathways. Fitzgerald (1989) has proposed that an axon reflex mechanism mediates inflammation in the joints. According to the hypothesis the damaged joint transmits neuronal impulses to the spinal cord where, via the axon reflex, signals are transmitted back to nerves at the joint that release pro-inflammatory substances which stimulate or exacerbate inflammation causing further stimulation of C-fibers. In addition postganglionic sympathetic fibers are thought to be stimulated which also release inflammatory substances.
The responses mediated by the peptides and transmitters released from sensory nerves include vasodilatation (via cGRP release), and increased vascular permeability (via SP release) (Jansco et al., 1967; Lembeck and Holzer, 1979; Saria, 1984; McDonald et al., 1996; Anichini et al., 1997; Strittmatter et al., 1997; Carlson et al., 1996; Lundeberg et al., 1996). In addition, the activation of the immune system initiates the attraction of white cells, activation of phagocytic function of neutrophils and macrophages, stimulation of the increased production and release of inflammatory mediators from these cells and the degranulation of mast cells and local release of histamine. (Helme and Andrews, 1979; Siato et al., 1986; Payan et al., 1984; Bar-Shavitz et al., 1980; Hartung et al., 1986; Johnson and Erdos, 1973; Naukkarinen et al., 1996). Substance P has been shown to stimulate the release of prostaglandin E2 and collagenase from cells in joints of patients with rheumatoid arthritis (Lotz et al., 1987), and further, induce the release of immune-active agents such as interleukin 1, tumor necrosis factor and interleukin 6 (Carleson et al., 1996). The result of this neuroendocrine cascade of events has been termed, neurogenic inflammation (Jancso, 1967) and works as a central network modulating the events between the immune, nervous and endocrine systems. A neurogenic disorder involves the release of neuropeptides, neuromodulators and other mediators of the neurogenic response including mediators from the immune nervous and endocrine systems.
There is significant and rapidly growing literature indicating that there is an important neurogenic component of inflammatory diseases, the prototypical example is rheumatoid arthritis (RA) (Heller et al., 1995; Anichini et al., 1997). Both autonomic and sensory afferent nerves have been implicated in the complex mechanisms involved in acute and chronic inflammation associated with RA (Levine et al., 1985; Jessel, 1985, Heller et al., 1995). Two neuropeptides, implicated as mediators of neurogenic inflammation, are Substance P (SP) and calcitonin gene related peptide (cGRP) (Anichini et al., 1997). Recent evidence obtained in a rat model for arthritis indicates that there is an increase in tissue content of the neuropeptides following adjuvant stimulation of joint inflammation in the rat (Ahmed et al., 1995). Additional studies support the role of SP and cGRP in mediating inflammation, leading to neuropathic pain, in which, levels of both are elavated when inflammation is induced (Anichini et al., 1997; Strittmatter et al., 1997; Carlson et al., 1996; Lundeberg et al., 1996). Also very recently pituitary adenylate cyclase-activating peptide (PACAP) has been added to this collection of peptide mediators of neurogenic inflammation and release from sensory nerves (C-fibres) (Wang et al., 1996)
The results of experiments performed in rat models for acute joint inflammation have demonstrated that local joint inflammation stimulated by articular administration of carrageenan is blocked by neurokinin (NK) receptor antagonists (the receptor responsible for binding SP) and the sensory neuron lesioning drug, capsaicin (Lam and Ferrel, 1989, Pierce et al., 1996). Capsaicin is currently used as a therapeutic treatment of inflammation but irritation and application as a topical agent limit the effectiveness. These observations are consistent with earlier observations implicating both of these peptides (SP and cGRP) in arthritis.
Both sensory and sympathetic nerves densely innervate the synovium and these neurons are known to release the neuropeptides SP and cGRP. The levels of the sensory neuropeptides SP and cGRP have been shown to be elevated in the synovium of RA patients. Although recent emphasis has been placed on these two peptides (SP+cGRP), there are a number of other pro-inflammatory mediators that may be involved in neurogenic inflammation such as but not limited to; PACAP, VIP, NK1, 5-HT, TNF, IL-1, IL-6, ACh, nitric oxide (NO), NGF and arachadonic acid.
In addition to the sensory afferent involvement in arthritis, there is also evidence that autonomic efferent input plays a role in acute inflammation and inflammatory disease (Heller et al. 1995; Janig, et al., 1997). Of particular interest are the results of experiments using experimental arthritis in the rat in which sympathetic denervation prevented the development of arthritis (Levine et al., 1986). Interestingly, rheumatoid arthritis patients without particular neurologic complaints showed a high proportion of neurophysiologic abnormalities, indicating the involvement of the sympathetic nervous system (Good et al., 1965).
Botulinum toxin has been shown to inhibit the release of all neurotransmitters and some peptides from efferent motor and autonomic nerves studied to date. It is very likely that the toxin can inhibit all substances that are released from cells by a vesicular mechanism when enough toxin is administered to enter these cells and antagonize vesicular release. This may also be accomplished by methods that temporarily increase the permeability of cells or by packaging the toxin in a form that more readily passes the membrane barrier. One example would be the use of lipid vesicles that would fuse to the membrane of the target cells and allow the toxins contained in these vesicles to enter the cells. As discussed above there is overwhelming evidence that the botulinum toxins inhibit neurotransmitter release by interfering with normal vesicular release mechanisms. Evidence exists that BoNTs do indeed inhibit the release of neuropeptides as evidenced by the data for inhibition of SP (Ashton and Dolly, 1988). Both SP and cGRP are both released from afferent sensory neurons that carry sensory information back to the central nervous system.
The properties of the metalloendopeptidase activity of the light chain of botulinum neurotoxins have been described above. A similar zinc-dependent metalloendopeptidase, neutral endopeptidase is responsible for the degradation of inflammatory mediators such as SP and CGRP (Nadel, 1994; Katayama et al., 1991; Nadel and Borson, 1991). Therefore, it is hypothesized by the inventor that BoNT acts enzymatically to cleave either proteins such as SNAP-25, syntaxin, and synaptobrevin, which are critical to vesicular release or direct cleavage of inflammatory mediators including but not limited to SP, CGRP, PACAP, VIP. The latter cleavage occurs within the cell or neuron and/or after this mediator is released into the extracellular environment. The long-term effects of the toxin are most likely related to the intracellular effects of the proteolytic activity of the toxin.
Arthritis for example, affects approximately 40 million Americans (1 in 7) and with the graying of America it is anticipated that this number will grow from 40 to 60 million by the year 2020. Of the 40 million about two thirds have moderate disease and about 10% are severely affected. Arthritis is the number one cause of disability in America and the estimated annual cost of arthritis is 55 billion dollars in medical care and indirect costs such as lost wages. There are more than 127 different types of arthritis as defined by the Arthritis Foundation. The etiology of arthritis is unknown however, the disease is characterized by chronic inflammation of the synovial joints. Over the last decade it has become increasingly more evident that arthritis, in particular, rheumatoid arthritis (RA) has a neurogenic component. In particular, the nervous system plays an important role in determining the pattern and severity of joint destruction therefore, the nervous system plays a crucial role in the pathophysiology of this chronic disease.
Synovitis has been referred to as the "first and truest kind of arthritis" with RA the prototypical example of this condition. This chronic inflammatory disease can, over many years, result in damage to the joint itself. There are other forms of arthritis that do not involve synovitis yet are characterized by inflammation such as ankylosing spondylitis, which is a genetic disorder and 7% of the US population has the gene (HLA-B27) and therefore, the potential to develop this disease. An estimate of 1% of the population suffers from this disease. Psoriatic arthritis is related to both synovitis and ankylosing spondylitis and can be distinguished by the joints that are affected including the general lack of symmetry of psoriatic joint involvement and the less prominent fatigue, tiredness and stiffness characteristic of rheumatoid arthritis.
In addition to arthritis, it has been proposed that neurogenic inflammation plays a fundamentally important role in the pathophysiology of migraine (Moskowitz, 1993). Working in much the same fashion as in arthritis, candidate mediators of migraine include vasoactive intestinal peptide (VIP), Substance P (SP) and calcitonin gene-related peptide (cGRP) (Goadsby et al., 1988, 1990; Goadsby and MacDonald, 1985; Goadsby and Shelley, 1990; Olesen 1991, 1994.) Another example whereby neurogenic inflammation takes place is in airway diseases, such as asthma, in which inflammatory mediators such as cGRP and SP play a major role in the inflammatory process (Barnes, 1996; Spina, 1996; Fisher et al., 1996).